ABSTRACT
Title of Thesis: CHARACTERIZATION OF LISTERIA
MONOCYTOGENES ISOLATED FROM
ORGANIC RETAIL CHICKEN
Emily T. Yeh, Master of Science, 2004.
Thesis directed by:Dr. Jianghong Meng
Department of Nutrition and Food Science
Listeria monocytogenes is an important foodborne pathogen. It is commonly found in the environment, frequently present in the gut of cattle, poultry, and pigs and can be transmitted to ready-to-eat foods as well as raw meat products. However, no data are available on the prevalence of L. monocytogenes in organic foods. In this study, 210 organic chickens collected from retail stores in the Washington DC area were examined for the presence of Listeria sp. using a modified Food and Drug Administration protocol developed to isolate the organism from meat products. Forty-eight organic chickens were positive for L. monocytogenes. The isolates were serotyped using PCR and subtyped by Pulsed-field gel electrophoresis (PFGE) to determine their genetic relatedness. The data revealed that several Listeria sp. were present on raw retail chicken and L. monocytogenes serotypes associated with human listeriosis were also identified in the product.
CHARACTERIZATION OF LISTERIA MONOCYTOGENES ISOLATED FROM RETAIL ORGANIC CHICKEN
by
Emily T. Yeh
Thesis submitted to the Faculty of the Graduate School of the
University of Maryland, College Park in partial fulfillment
of the requirements for the degree of
Master of Science
2004
Advisory Committee:
Associate Professor Jianghong Meng
Associate Professor Mark Kantor
Assistant Professor Liangli Yu
Acknowledgements
I would like to thank all my friends, family and collegues who have supported me in my decision to attend graduate school as well as those who have taken the time to listen, discuss and argue about almost everything else in my subsequent years of graduate school. Thank you for helping me maintain my sense of mental balance and humor.
Table of Contents
List of Tables…………………………………….……………………….……iv
List of Figures………………………………………………….……..………..v
Introduction……………………………………………………………………1
Organism………………………………………………………..………..2
Virulence………………………………………………….…….……….3
Disease……………………………………………………….….………..7
Characterization……………………………………………..…...…..11
Listeria and Food………………………………………………………16
Materials and Methods……………………………..…………...………….23
Sample Collection and Preparation……………...…………………23
Bacterial Isolation……………………………….…………………..23
Isolation Confirmation……...…………………..……………………25
Serotyping by PCR…..…………………………..……………………..26
PFGE analysis……….……………………….………………………...29
Results…...…………………………………………….………………………32
Overall Prevalence…………….…………………………………….32
Organic Chicken Prevalence………………….……………………..33
Conventional Chicken Prevalence………………………………….33
Rapid L.mono Confirmation…………………………………………..34
Serotyping………………………..…………….………………………35
Subtyping…………………………..…………..……………………….38
Discussion……………………………………………...……………………….41
Conclusion………………………………………….…………………………46
References………………………………………..……………………………48
List of Tables
Table 1: L. monocytogenes genomics…………………...…………………….3
Table 2: The genes that play a role in the pahtogenesis of L.
monocytogenes………………………………………..………………….5
Table 3: Comparison of mortality and hospitalization rates among L.
monocytogenes and other common foodborne pathogens……...…..9
Table 4: Summary of historical listeriosis outbreaks and their vehicle
of transmission…………………………………………………………17
Table 5: Prevalence of L. monocytogenes on chicken carcasses from
other studies performed worldwide……………….………………..20
Table 6: Summary of PCR primers used to serotype……………...………29
Table 7: Summary of L. monocytogenes results for organic and
conventional chickens by category………………………………...34
Table 8: Summary of serotypes in organic chicken…………..…………..37
Table 9: Summary of serotypes in conventional chicken…….……….…37
List of Figures
Figure 1: Virulence gene cluster located in L. monocytogenes………….4
Figure 2: Intracellular invasion by L. monocytogenes……………………7
Figure 3: Overview of PCR primers used to serotype……………………..27
Figure 4: Confirmation of Listeria spp. on Rapid L.mono plates…...…….35
Figure 5: Example of all positive results for each of the serotype
primers………………………………………………………………….36
Figure 6: Example of PFGE patterns from some of the samples
examined………………………………………………………………..38
Figure 7: Dendrogram of PFGE isolates………………………...…….…...40
iv
Introduction
Listeriamonocytogenes, was first reported in 1924 by E.G.D. Murray [54]. Murray isolated the organism, which caused monocytosis, from rabbits and guinea pigs. In 1929, Nyfeldt described the Bacterium monocytogenes hominis [57] as pathogenic to humans as well. It was not until the early 1980’s that L. monocytogenes was first recognized as a food-borne pathogen [74].
Although today there is a clearer understanding of the organism and its relationship to other organisms, this has not always been the case. Throughout its history Listeria has been observed, studied and phylogenetically classified by numerous researchers. Because of the uncertainty of its phylogenetic position and its morphological similarity to the group of coryneform bacterium, names such as, Corynebacterium parvulum [79] and Corynebacterium infantisepticum [64] have been used to describe the organism.
It was not until the 1970s that its phylogenetic relationship, reinforcing its distinctiveness from the coryneform bacteria, was better understood. Its position was better clarified because of the development of numerical taxonomy, chemotaxonomy, DNA/DNA hybridization and rRNA sequencing techniques [70]. Currently the genus is taxonomically classified with its phylogenetic position being closely related to Brochothrix [20]. However its relationship with other low G+C% Gram-positive bacteria [26, 69, 87], as well as Bacillus and Staphylococcus still needs to be clarified.
Since the organism was first recognized, much information on its ecology, pathogenicity and the epidemiology of listeriosis has been revealed, yet the organism is still not completely understood nor is its presence in food under control.
Organism
Listeria is a gram-positive, non-sporulating, catalase positive, oxidase negative rod, which measures 0.5 um in diameter and 1 - 2 um in length. Gram stains show that the cells can be found in chains or as single rods. Growth of the organism on bacteriological media is enhanced by the presence of glucose or other fermentable sugars but is also dependent on the atmosphere and temperature in which they are grown. The organism can grow over a wide range of pHs (4.3-9.6), water activity (~ 0.83) and salt concentrations (up to 10 %) as well [83]. Listeria are aerobic, microaerophilic and facultatively anaerobic and can be cultured over a wide temperature range.
The organism has a growth temperature range of approximately 1C - 45C, [44], making it a psychrotroph and a mesophile. There are however, growth factors which are temperature dependent. For example, at 20-25C peritrichous flagella are formed and cause the organism to be motile, whereas at 37C the organism is weakly or non-motile [29]. Additionally, its ability to not only survive but to grow as a psychrotroph at 4C makes this pathogen unique from other commonly found food-borne pathogens which are usually inhibited from growth at refrigeration temperatures.
For many years the genus Listeria only contained one species, L. monocytogenes. Currently however, there are six recognized species including L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, and L. grayi [70, 93]. Although there are six distinct species they all have similar genetic homology which helps explain their similar phenotypic traits. Hemolysis as well as acid production are key characteristics in distinguishing among the species. At present only strains of L. monocytogenes are pathogenic to humans and animals, while L. ivanovii are only pathogenic in animals, particularly ruminants [95].
Virulence
The L. monocytogenes genome is approximately 3.0 Mb [53] (Genbank/EMBL accession number AL591824) and information on its sequence can be found at The Institute for Genomic Research ( Virulence and virulence-like genes on Listeria’s chromosome code for surface and secreted proteins as well as other regulators which help it to adapt to diverse environments and for expression of virulence traits (Table 1).
Table 1: L. monocytogenes genomics.
L. monocytogenesSize of chromosome (kb) / 2,944,528
G+C content (%) / 39
G+C content of protein-coding genes (%) / 38
Total number of protein-coding genes / 2853
Adapted from [35]
Further, species such as L.innocua lack genes which are essential for virulence. For example, a virulence gene such as one which codes for a surface protein and plays a role in invasion is present in L. monocytogenes but is absent in L.innocua and this may help explain the different pathogenic potentials of different species [35, 45].
Listeria monocytogenes and L. innocua both contain a virulence gene cluster located on an 8.2 kb pathogenic island on its genome [45] which is regulated by the main positive regulatory factor A regulon (PrfA) [18]. The cluster is located between the prs and ldh genes on the chromosome [36] (Figure 1).
Figure 1: Virulence gene cluster located in L. monocytogenes.
Modified from http://mcb.berkeley.edu/courses/mcb103/02PrfAModelSlide.gif
This cluster is the only one known to date that is involved in the virulence of Listeria. It contains the majority of the known virulence genes which are involved in the invasion and intracellular cycle of the pathogen. The cluster encodes six genes, prfA, plcA, hly, mpl, actA, plcB, and three additional small open reading frames (orfs) X, Y, and Z downstream of plcB [46]. The PrfA gene is essential for the virulence of L. monocytogenes. It acts as the master regulator of virulence and virulence-like genes to varying degrees [19]. The remaining virulence genes on the virulence gene cluster result in the protein products of listeriolysin O (LLO) by hly, a phosphatidylinositol-specific phospholipase C (PI-PLC) by plcA, a phosphatidylcholine-specific phospholipase C (PC-PLC) by plcB, a metalloprotease (mpl), an actin polymerization protein (ActA) by actA, and three genes, X, Y, and Z which functions are currently unknown.
Several other genes involved in virulence are located outside of but are still related to the gene cluster [46]. These genes which are involved in the production of surface proteins necessary for internalization of the pathogen to the host cell, include the inlA, inlB and inlC genes which code for internalin A, B, and C respectively as well as the iap virulence-like gene which codes for p60. However, it seems there are still other virulence and virulence-like genes which are either only partially regulated by or totally independent of PrfA [49, 84] (Table 2).
Table 2: The genes that play a role in the pathogenesis of L. monocytogenes. [68]
Gene / FunctionprfA / positive regulatory factor A / transcriptional activator
plcA / phosphatidylinositol-specific phospholipase C / aids in escape from vacuoles
plcB / phosphatidylcholine-specific phospholipase C / aids in escape from vacuoles
hlyA / listeriolysin O / escape from vacuoles
mpl / zinc-dependent metallooprotease / maturation of PlcB
actA / actin-polymerizing protein / cell-to-cell spread
inlA / internalin A / internalization
inlB / internalin B / internalization
Listeria’s pathogenicity is not only contributed by its existence as an intracellular pathogen but also to its ability to invade and replicate within a wide range of mammalian cells. The rate of internalization is dependent on the cell type and may be mediated by at least one bacterial surface protein. The bacterial surface proteins inlA and inlB [10] are responsible for promoting the binding and internalization by either E-cadherin or the Met receptor tyrosine kinase and PI3-kinase activation respectively [21].
Once the organism is internalized a key virulence determining protein, LLO, as well as other secretory proteins PI-PLC and PC-PLC, aid the release of the bacteria from the vacuole which it resides in [31]. After cells have multiplied in the cytosol, an actin-based motility protein necessary for cell-to-cell spread, ActA is synthesized. The protein induces the polymerization of host actin filaments and allows the pathogen to propel itself into other cells as a type of pseudo-pod while evading the host’s defenses [63]. This intracellular cycle and cell-to-cell spread is then continued onto the next cells to continue the infection. (Figure 2).
Figure 2: Intracellular invasion by L. monocytogenes [89].
Disease
Listeriosis is the disease caused by L. monocytogenes infections. Listeria is widely distributed in the environment and can also be found in the gastrointestinal tract of individuals who remain as asymptomatic carriers. This non-invasive listeriosis occurs in healthy adults but generally only amounts to gastrointestinal illness, fever, vomiting and diarrhea, where the degree of severity is dependent on the characteristics of the host and the organism’s environment.
On the other hand, the more severe form of listeriosis is invasive listeriosis. The most common invasive listeriosis infections occur in children, the elderly, pregnant women and their fetuses and the immunocompromised. With the onset of epidemics such as HIV/AIDS, there has been an increase in the size of the population at risk of morbidity and mortality due to this type of listeriosis [75]. The disease can manifest as septicemia, meningitis, meningoencephalitis or febrile gastroenteritis and can cause still births and abortions [93].
The infective dose has not yet been definitively determined but it may take less than 1000 cells to cause infection. However, this is dependent on the immunity of the infected individual and the strain of the organism. The incubation period can range from a few days to three weeks and may be preceeded by gastrointestinal symptoms which manifest after approximately 12 hours incubation [52].
The rate of normal healthy adult infection from listeriosis is low. There are approximately 0.7 cases per 100,000 persons. However, the infection is more common in children at a rate of 10 cases per 100,000 person, and the elderly with 1.4 cases per 100,000 person [32]. Pregnant women are seventeen times more likely than healthy adults to acquire the infection [86].
Although listeriosis is considered a food-borne infection and most outbreaks are transmitted by food, there have been reports of large outbreaks attributed to other modes of transmission. For instance, a neonatal outbreak in Costa Rica involved the use of contaminated mineral oil for cleaning infants after delivery [77].
In addition the pathogens which cause neonatal bacterial meningitis in North America, Listeria is the third most common pathogen followed by group B streptococcus and E. coli [23]. Fetuses can acquire the infection through the mother who has either colonized the organism in the gastrointestinal (GI) tract after consumption of contaminated foods or during childbirth if a mother is carrying Listeria in the GI or the perianal region, which in turn can contaminate the skin and respiratory tract of the child during birthing. Although cases unrelated to food do occur, foodborne transmission is the most common source of transimission of Listeria to humans.
In the U.S there are an estimated 76 million cases of food-borne illnesses each year. The incidences of listeriosis only average 2500 infections yearly but cause 500 fatalities [52]. Although the actual number of infections is low, a mortality rate, which can be as high as 20-30% regardless of antimicrobial treatment shows the danger that the presence of Listeria poses in foods. The mortality rate is considerably higher than the more common infections from other food-borne pathogens such as Escherichia coli O157:H7 (E. coli), Campylobacter spp. and Salmonella spp. [52] (Table 3).
Table 3: Comparison of the mortality rate among L. moncytogenes and other common food-borne pathogens [52].
Pathogen / Mortality Rate (%) / Hospitalization Rate (%) / Total CasesCampylobacter spp / <1 / 17.3 / ~1.9 million
E. coli O157:H7 / <1 / 3.0 / ~62,500
Salmonella spp. / <1 / 25.6 / ~1.3 million
L. monocytogenes / 20.0 / 3.8 / 2500
There have been several sporadic and epidemic outbreaks worldwide implicating Listeria contaminated foods (Table 4) [1, 3, 4, 16, 27, 74, 81]. Foods which are denoted as ready to eat (RTE) foods (deli meats, salads etc.), unpasteurized dairy foods (cheese and milk), cured and raw meats (hot dogs, undercooked chicken), and items such as prepared seafood salads and even raw and unprocessed meats have been common foods implicated [75]. Although a look back at some initial recordings of the organism and their related outbreaks demonstrate that a food-to-human route of transmission was likely, it was not established until the 1980’s [73]. There is still more to uncover concerning Listeria’s relationship between environment, human and food.
Listeriosis is often treated with antimicrobials. The most favored treatment consists of using a combination of ampicillin and an aminoglycoside [42]. However the use of vancomycin in place of ampicillin is acceptable as well. Another treatment is the use of trimethoprim-sulfamethoxazol (TMP-SMZ) and rifampin. Cephalosporins, which are typically used for treatment of meningitis, are not effective for treating listeriosis because of L. monocytogenes resistance to this drug. However cephalosporins can be used in combination with ampicillin for listerial meningitis [75]. Overall, antimicrobial treatment against listeriosis can be slow and may even be untreatable or persistant [85].
However remaining vigilant about monitoring the pathogen is also important. This can be demonstrated by examining the way outbreaks are analyzed. PFGE patterns show which strains are likely responsible for an outbreak by matching the genomic patterns from clinical samples to suspected foods which may be vehicles of transmission. With the cooperation and standardization of laboratories performing the analysis, this becomes a rapid method for determining the source of infection by sharing of information and perhaps limiting the number of infections and subsequent deaths [38].
Characterization of the Organism
Phenotyping and genotyping methods to discriminate bacterial strains are valuable tools which have different levels of discriminatory power. These methods are able to provide information on strains which may be responsible for an outbreak, or to identify the relationship between isolates implicated in an outbreak, and also to help determine a source of transmission for an outbreak.
Serotyping is based on the antigenic determinations expressed on the cell surface of the organism. These antigens are produced by lipoteichoic acids, membrane proteins, and extracellular organelles such as fimbriae and flagella [82]. Different strains of L. monocytogenes express different antigenic determinations, thus each strain can be serologically identified.
Listeria strains are separated based on flagellar (H) and somatic (O) antigens resulting in more than 13 serotypes [82, 38]. Genotypic analysis generally group Listeria into two main lineages, Lineage I and II while it is believed that there may be a third subgroup, Lineage III, as well.
Lineage I includes serotypes 1/2a, 1/2c, 3a, and 3c 1, Lineage II includes serotypes 1/2b, 3b, 4b, 4d and 4e while serotypes 4a and 4c have been grouped with the third less common Lineage III [66, 37, 13]. The three serotypes which most commonly cause disease (> 95%) are 1/2a, 1/2b and 4b [60]. Although serotype 4b is most frequently implicated in foodborne diseases, it is serotype 1/2a which is most frequently isolated from foods [34].
Historically, serotyping of Listeria by antiserum has shown to be unreliable. A study by [76] and the World Health Organization (WHO), demonstrated that different laboratories using antiserum serotyping methods, either could not 100% correctly identify or 100% agree on the correct serotype of all the given isolates [76]. In addition, serotyping with antiserum has been shown to have less discriminating power than other methods.
For example, a Mismatch Amplification Mutation Assay (MAMA) using mismatched PCR primers targeting different sites in the hly gene was developed to rapidly screen L. monocytogenes isolates into their respective phylogenetic divisions [43]. Other methods such as one using PCR primers which target select sequences in the four major serotypes to adequately differentiate them have also been recently developed. The results obtained with the PCR primers agree significantly enough with the traditional slide agglutination method [9] that perhaps this can one day be a standardized method.